Apoptosis is a cell suicide process involved in various physiological and pathological activities, such as embryonic development, cancer and autoimmune diseases. Induction of apoptosis can be mediated through both caspase dependent and independent processes. The mechanisms of caspase-independent apoptosis have not been well understood. AIF is a mitochondrial flavoprotein that triggers caspase-independent apoptosis. We have cloned a novel AIF homologous molecule designated as AMID. AMID is localized to the outer membrane of mitochondria. Overexpression of AMID induces caspase-independent apoptosis. Moreover, AMID is induced by the tumor suppressor p53 and expression of AMID is down-regulated in tumors in comparison to their individually matched normal tissues. We hypothesize that AMID induces caspase-independent apoptosis through novel mechanisms and AMID is a tumor suppressor involved in p53-mediated downstream effects. To test our hypotheses, we have proposed three specific aims: 1). To investigate the mechanisms of AMID-induced caspase-independent apoptosis; 2). To investigate the roles of AMID in p53-mediated apoptosis and cell growth arrest; 3). To determine whether AMID is a tumor suppressor gene using in vitro cell culture systems and in vivo gene knock-out studies in mice. Successful completion of the proposed studies will help to understand the molecular mechanisms of caspase-independent apoptosis and roles of AMID in p53-mediated biological effects and tumorigenesis.